AOAC Official Method 2000.13 Reveal for E
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D38B40ED62B34A2CA164AD5D81A644CD |
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0.03 |
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2 |
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日期： |
2024-7-30 |
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17.4.06,AOAC Official Method 2000.13,Reveal for E. coli O157:H7 Test System,in Selected Foods,8-Hour Method,First Action 2000,(Applicable only to the detection of E. coli O157:H7 in raw,ground beef, raw beef cubes, and iceberg lettuce rinse.),See Table 2000.13 for the results of the interlaboratory study supporting,acceptance of the method.,A. Principle,Reveal 8 uses a proprietary enrichment medium (8-Hour medium),and a rapid lateral flow immunoprecipitate test Device to yield a result,in just over 8 h total test time. The 8-Hour medium provides E.,coli O157:H7 with readily available nutrients and other factors required,for its survival and rapid growth to a detection level by the,Device in only 8 h. The Device contains antibodies with high specificity,to E. coli O157:H7 antigens. These antibodies are bound to,colloidal gold and, separately, to a solid support matrix.Aportion of,the test enrichment is placed into the sample port of the Device initiating,flow. Any E. coli O157:H7 antigen present will bind to the,gold-conjugated antibodies forming an antigen–antibody–,chromogen complex. This complex flows across the lateral,flow membrane and is subsequently bound by antibody immobilized,on the membrane causing the gold conjugate to precipitate forming a,visible line and indicating a positive reaction. Proper test completion,and flow are indicated by a control line which forms higher up in the,test window, verifying a valid test run. Absence of a control line invalidates,the test.,B. Media and Reagents,(a) 8-Hour medium for E. coli O157:H7, unitized (one test per,bottle).—Empty contents of one bottle into sterile container. Add,225 mL sterile water at 43EC. Mix.,(b) Device.—One test per device.,(c) Butterfield’s phosphate buffer (BPB) diluent.—Mix 34.0 g,monopotassium phosphate in 500 mL distilled water. Adjust pH to,7.2 using 1M NaOH (40 g/L). Dilute to 1 L with distilled water. Autoclave,for 15 min at 121EC. Add 1.25 mL to sterile 1 L flask and dilute,to 1 L with sterile water.,(d) Diagnostic reagents.—Necessary for culture confirmation of,presumptive positive tests. See chapter on E. coli and coliform bacteria,in BAM or FSIS Laboratory Communication No. 38.,(e) Hemorrhagic coli (HC) agar.—Prepare as follows: tryptone,20.0 g; bile salts No. 3, 1.12 g; NaCl, 5.0 g; sorbitol, 20.0 g;,4-methylumbelliferyl-b-D-glucuronide (MUG) reagent, 0.1 g;,bromocresol purple, 0.015 g; agar, 15.0 g; and distilled water, 1.0 L.,Dissolve ingredients in distilled water by heating with stirring. Autoclave,15 min at 121°C. Final pH 7.2 ± 0.2.,(f) Biosynth Chromogenic medium (BCM) O157:H7(+),agar.—Commercially available from Biosynth International, Inc.,(1665 W. Quincy Ave, Suite 155, Naperville, IL 60504, USA). Prepare,as follows: BCM O157:H7(+), 80.0 g; N,N-dimethylformamide,(DMF, Sigma, Cat. No. D4254), 5.0 mL; sodium novobiocin (0.2%,solution, Sigma, Cat. No. N1628), 5.0 mL; potassium tellurite (0.1%,solution, Sigma, Cat. No. P0677), 0.2 mL; and distilled water, 1 L.,Add DMF to distilled water followed by BCM powder and heat to,boiling to dissolve. Adjust pH to 6.8 ± 0.1. Do not autoclave. Cool to,50–55°Cin a water bath. Separately filter sterilize novobiocin and potassium,tellurite and aseptically add to media.,C. Apparatus,(a) Incubators.—Maintaining 35–37EC and 42–43EC.,(b) Water bath.—Maintaining 100 ± 5EC.,(c) Syringe.—Equipped with 0.45 mm or smaller porosity filter.,(d) Test tubes.—16 ′ 100 mm borosilicate glass.,(e) Pipettor.—Accurately dispensing 0.12 mL.,(f) Graduated cylinder.—250 mL capacity with 5 mL graduations.,(g) Stomacher bag.,D. Preparation of Test Suspensions,For iceberg lettuce, aseptically shred lettuce and transfer 50 g to,sterile bag. Add 50 mL BPB diluent, B(c), close bag, and shake by,hand for 15 min. Aseptically decant 25mLinto 500mLscrew-capped,Erlenmeyer flask containing 225 mL 8-Hour medium, B(a). Cap,loosely, mix by shaking lightly, and incubate 8 h at 42–43EC. Remove,5mLculture and place in test tube,C(d). Place tube in water bath,C(b),at 100EC for 10 min. Cool to room temperature.,For raw beef cubes and raw ground beef, aseptically weigh 25.0 g,test portion into stomacher bag,C(g), containing 225mL8-Hour medium,B(a). Mix well by hand-kneading and incubate 8 h at 42–43EC.,Remove 5 mL and place in test tube, C(d). Place tube in water bath,C(b), at 100EC for 10 min. Cool to room temperature.,E. Test Procedure,Open pouch containing one Device, B(b). Use one Device per,test. Transfer 0.12 mL heat-killed culture from test tube, prepared as,in D, into sample port of Device. Incubate 15 min at room temperature.,F. Reading Results,Examine Device, B(b), immediately after ……
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